Cell flow apparatus and method for real-time of cellular responses

• US 6,280,967 B1
• Filed: 08/05/1999
• Issued: 08/28/2001
• Est. Priority Date: 08/02/1996
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Axiom Biotechnologies Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Okun, Alex, Okun, Ilya, Ransom, John
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/370,786
• Time in Patent Office: 754 Days
• Field of Search: 435/29, 435/34, 435/283.1, 435/968, 435/286.1
• US Class Current: 435/29
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5076 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• CPC Class Codes: G01N 33/5079 Mitochondria
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 35/00 Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

An apparatus and method for real-time measurement of a cellular response of a test compound or series of test compounds (303) on a flowing suspension of cells (349), in which a homogeneous suspension of each member of a series of cell types (349) is combined with a concentration of a test compound (303), directed through a detection zone (355), and a cellular response of the living cells is measured in real time as the cells in the test mixture are flowing through the detection zone (355). The apparatus may be used in automated screening of libraries of compounds, and is capable of real-time variation of concentrations of test and standard compounds and generation of dose/response profiles within a short time span.

Claims

1. A method for identifying compounds which produce a cellular response comprising:

(a) using an automated and computer controlled apparatus to sequentially combine each of a plurality of cell suspensions with one or more test compounds to sequentially form each of a plurality of test mixtures;

(b) sequentially directing each of said plurality of test mixtures through a detection zone, the detection zone being capable of detecting a plurality of cellular responses simultaneously; and

(c) simultaneously measuring a plurality of cellular responses to the one or more test compounds in said suspended cells as each of said plurality of test mixtures are flowing through said detection zone.

2. The method of claim 1, wherein said plurality of cellular responses includes a cellular response selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, activation or inhibition of cellular necrosis, and cellular toxicity.

3. The method of claim 1, wherein said plurality of cellular responses are measured by contacting said cells with one or more response indicating reagents which generate signals indicative of particular cellular responses.

4. The method of claim 3, wherein said signal generated by said response indicating reagents is fluorescence.

5. The method of claim 1, wherein said step of sequentially directing each of said plurality of test mixtures through a detection zone comprises directing each of said plurality of test mixtures through a detection zone which is capable of detecting said cellular responses in individual cells.

6. The method of claim 1, wherein said step of sequentially directing each of said plurality of test mixtures through a detection zone comprises directing each of said plurality of test mixtures through a flow cytometer.

7. The method of claim 1, wherein the step of simultaneously measuring a plurality of cellular responses comprises measuring said plurality of cellular responses in said suspended cells as a slug is flowing through said detection zone.

8. The method of claim 1, wherein each of said plurality of cell suspensions comprises more than one type of cell.

34. The method of claim 1, further comprising the steps of:

(d) combining a suspension of said cells with one or more standard compounds having a known effect on said cellular response of said cells to form standard mixtures;

(e) directing the standard mixtures through the detection zone; and

(f) measuring the cellular response of said cells to said one or more standard compounds.

35. The method of claim 1, wherein one or more standard compounds and said one or more test compounds are simultaneously mixed with each of said cell suspensions, and said measuring step detects whether said one or more test compounds alter a known effect of said one or more standard compounds.

36. The method of claim 35, wherein said one or more standard compounds is an antagonist of said cellular response.

37. The method of claim 35, wherein said one or more standard compounds is an agonist of said cellular response.

38. The method of claim 35, wherein said cell suspensions comprise more than one cell type.

39. The method of claim 35, wherein the step of directing said test mixtures through a detection zone comprises directing said test mixtures through a flow cytometer.

72. The method of claim 1, wherein a range of concentrations of said test compounds is combined with said cell suspensions.

73. The method of claim 1, further comprising combining one or more compounds which produce a known cellular response with said plurality of cell suspensions and said test compounds.

74. The method of claim 73, wherein a range of concentrations of said one or more compounds which produce a known cellular response is combined with said plurality of cell suspensions and said test compounds.

78. The method of claim 1, wherein a range of concentrations of said agents capable of binding said molecules is combined with said samples.

9. A method for identifying compounds which produce a cellular response comprising:

(a) using an automated and computer controlled apparatus to sequentially combine each of a plurality of cell suspensions with one or more test compounds to form each of a plurality of test mixtures;

(b) sequentially directing each of said plurality of test mixtures through a detection zone, said detection zone being capable of detecting cellular responses in individual cells; and

(c) measuring one or more cellular responses to said one or more test compounds in individual suspended cells as each of said plurality of test mixtures are flowing through said detection zone.

10. The method of claim 9, wherein said one or more cellular responses is selected from the group consisting of activation or inhibition of a receptor mediated response, activation or inhibition of an ion channel, activation or inhibition of a non-selective pore, activation or inhibition of a second messenger pathway at a point downstream of a receptor or channel, activation or inhibition of apoptosis, and activation or inhibition of cellular necrosis, and cellular toxicity.

11. The method of claim 9, wherein said one or more cellular responses is measured by contacting said cells with one or more response indicating reagents which generate signals indicative of particular cellular responses.

12. The method of claim 11, wherein said signal generated by said response indicating reagents is fluorescence.

13. The method of claim 9, wherein said step of directing each of said plurality of test mixtures through a detection zone comprises directing each of said plurality of test mixtures through a flow cytometer.

14. The method of claim 9, wherein each of said plurality of cell suspensions comprises more than one type of cell.

15. The method of claim 9, wherein the step of simultaneously measuring a plurality of cellular responses comprises measuring said plurality of cellular responses in said suspended cells as a slug is flowing through said detection zone.

75. The method of claim 9, wherein a range of concentrations of said test compounds is combined with said cell suspensions.

76. The method of claim 9, further comprising combining one or more compounds which produce a known cellular response with said plurality of cell suspensions and said test compounds.

77. The method of claim 76, wherein a range of concentrations of said one or more compounds which produce a known cellular response is combined with said plurality of cell suspensions and said test compounds.

16. A method of determining whether a sample contains one or more molecules comprising:

using an automated and computer controlled apparatus to sequentially combine one or more agents capable of binding one or more molecules with each of a plurality of samples to form a plurality of test mixtures;

sequentially directing each of said plurality of test mixtures through a detection zone; and

determining whether said one or more agents are bound to said one or more molecules as each of said test mixtures are flowing through said detection zone.

17. The method of claim 16, wherein said one or more agents are fixed to a solid support.

18. The method of claim 17, wherein a plurality of agents capable of binding a plurality of molecules are fixed to uniquely identifiable solid supports.

19. The method of claim 18 wherein said determining step comprises detecting a signal from an individual solid support wherein said signal indicates that said agent on said solid support has bound to said molecule.

20. The method of claim 16, wherein said one or more agents are selected from the group consisting of antibodies, nucleic acids, polypeptides, enzymatic substrates, and receptors.

21. The method of claim 16, wherein said one or more molecules are selected from the group consisting of polypeptides, nucleic acids, receptor ligands, enzymatic agonists and enzymatic antagonists.

22. A method of determining whether a sample contains one or more polypeptides comprising:

attaching one or more antibodies which specifically bind said one or more polypeptides to solid supports;

using an automated and computer controlled apparatus to sequentially combine said one or more antibodies attached to solid supports with samples to form a plurality of test mixtures;

sequentially directing each of said plurality of test mixtures through a detection zone; and

determining whether said one or more antibodies are bound to said one or more polypeptides as each of test mixtures are flowing through said detection zone.

23. The method of claim 22, wherein a plurality of antibodies which specifically bind to a plurality of polypeptides are fixed to uniquely identifiable solid supports.

24. The method of claim 23 wherein said determining step comprises detecting a signal from an individual solid support wherein said signal indicates that said antibodies have bound to said molecules.

25. The method of claim 24, wherein said signal is generated by detectably labeled secondary antibodies which bind said molecules.

26. A method of determining whether a sample contains one or more nulceic acids comprising:

attaching one or more nucleic acid probes which specifically bind said nucleic acids to solid supports;

using an automated and computer controlled apparatus to sequentially combine said one or more probes attached to said solid supports with samples to form a plurality test mixtures;

sequentially directing each of said plurality of test mixtures through a detection zone; and

determining whether said one or more probes are bound to said one or more nucleic acids as each of said test mixtures are flowing through said detection zone.

27. The method of claim 26, wherein a plurality of nucleic acid probes are fixed to uniquely addressable solid supports.

28. The method of claim 27, wherein said determining step comprises detecting a signal from an individual solid support wherein said signal indicates that said probes have bound to said nucleic acids.

29. The method of claim 28, wherein said signal is generated by detectable nucleic acid probes which bind said nucleic acids.

30. A method of determining whether a sample contains one or more single nucleotide polymorphisms comprising:

performing an extension reaction on a each of a plurality of nucleic acid samples using one or more nucleic acid primers for each of said plurality of nucleic acid samples, said one or more nucleic acid primers having a 3′

end immediately adjacent to the polymorphic base of one or more single nucleotide polymorphisms, thereby incorporating one base into said nucleic acid primers;

attaching the extended primers to solid supports;

sequentially directing said solid supports having primers attached thereto for each of said plurality of nucleic acid samples through a detection zone in an apparatus; and

determining the identities of the bases incorporated in said extension reaction.

31. The method of claim 30, wherein a plurality of primers are fixed to uniquely identifiable solid supports.

32. The method of claim 31, wherein said determining step comprises detecting a signal from an individual solid support wherein said signal is indicative of the identity of the polymorphic base in a SNP.

33. The method of claim 32, wherein said signal is generated by detectably labeled nucleotides incorporated in said extension reaction.

40. A method of obtaining cells having a desired phenotype or cellular response profile comprising the steps of:

(a) using an automated and computer controlled apparatus to sequentially combine a each of a plurality of cell suspensions with one or more compounds which produce a cellular response to form a plurality of test mixtures;

(b) sequentially directing each of said plurality of test mixtures through a detection zone, said detection zone being capable of detecting a plurality of cellular parameters simultaneously;

(c) simultaneously measuring a plurality of cellular parameters in said suspended cells as each of said plurality of test mixtures are flowing through said detection zone; and

(d) delivering cells having a desired phenotype or cellular response profile into a receptacle.

79. The method of claim 40, wherein a range of concentrations of said compounds is combined with said cell suspensions.

80. The method of claim 40, further comprising combining one or more compounds which produce a known cellular response with said plurality of cell suspensions and said compounds.

81. The method of claim 80, wherein a range of concentrations of said one or more compounds which produce a known cellular response is combined with said plurality of cell suspensions and said compounds.

41. A method of obtaining cells having a desired phenotype or cellular response profile comprising the steps of:

(a) using an automated and computer controlled apparatus to sequentially combine a each of a plurality of cell suspensions with one or more compounds which produce a cellular response to form a plurality of test mixtures;

(b) sequentially directing each of said plurality of test mixtures through a detection zone, said detection zone being capable of detecting cellular responses in individual cells;

(c) simultaneously measuring a plurality of cellular parameters in said suspended cells as each of said plurality of test mixtures are flowing through said detection zone; and

(d) delivering cells having a desired phenotype or cellular response profile into a receptacle.

82. The method of claim 41, wherein a range of concentrations of said compounds is combined with said cell suspensions.

83. The method of claim 41, further comprising combining one or more compounds which produce a known cellular response with said plurality of cell suspensions and said compounds.

84. The method of claim 83, wherein a range of concentrations of said one or more compounds which produce a known cellular response is combined with said plurality of cell suspensions and said compounds.

42. An apparatus comprising:

an automated test compound source capable of sequentially providing each of a plurality of test compounds;

an automated test substrate source capable of sequentially providing each of a plurality of test compounds;

a mixing chamber in fluid communication with said test compound source and said test substrate source, wherein said mixing chamber is adapted to combine a test compound received from said test compound source with a test substrate received from said test substrate source to generate a mixture; and

a detector in fluid communication with said mixing chamber, said detector being capable of detecting an interaction between said test compound and said test substrate while said mixture is passing through said detector.

43. The apparatus of claim 42, wherein:

said test compound source comprises one or more sample inputs for sequentially providing each of a plurality of samples, each of said samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;

said test substrate source comprises one or more cell or particle inputs for sequentially providing a each of a plurality of cell suspensions or particles;

said mixing chamber receives the samples from said test compound source, receives the cell suspensions or particles from the cell or particle input and mixes each sample with the cell suspension or particles; and

said detector is capable of detecting a plurality of signals simultaneously such that said detector can simultaneously measure a plurality of cellular responses in the suspended cells or simultaneously determine whether a plurality of molecules are present in said samples.

44. The apparatus of claim 43 wherein said detector detects a signal from a single cell in said cell suspension or from a single particle.

45. The apparatus of claim 43 wherein said detector is a flow cytometer.

46. The apparatus of claim 43, further comprising a coupler disposed between said mixing zone and said detector.

47. The apparatus of claim 46, wherein said coupler delivers slugs comprising said samples and said cell suspension or particles to said detector.

48. The apparatus of claim 47, wherein said coupler delivers substantially undiluted slugs to said detector.

49. The apparatus of claim 43, wherein said detector delivers cells having a desired phenotype or cellular response to a receptacle.

50. The apparatus of claim 43 wherein said one or more sample inputs comprise an automated robotic sampler capable of sequentially selecting each of a plurality of specified test compounds from a library of test compounds.

51. The apparatus of claim 50 further comprising:

a controller, coupled to said one or more sample inputs, for controlling the operation of the automated robotic sampler; and

a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by said one or more sample inputs from said test compound library.

52. The apparatus of claim 51 further comprising a gradient pump having an input and an output, coupled to said sample input, for adjusting the concentration level of said test compound transferred to said mixing zone from said sample input, wherein:

said sample input comprises;

a first intake nozzle for receiving each of the said specified test compounds;

a second intake nozzle for receiving a buffer solution; and

wherein said gradient pump is coupled to the first and second intake nozzles and receives specified concentrations of each of the specified test compounds by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.

53. The apparatus of claim 52 further comprising a second pump, coupled to an input of said mixing zone, for pumping said suspension of cells or said particles from said cell or particle input into the mixing zone.

54. The apparatus of claim 53 further comprising a reaction developing line, having an input coupled to an output of said mixing zone and an output in fluid communication with said detector wherein said reaction developing line provides a reaction time delay for a mixture received from the mixing zone.

55. The apparatus of claim 54 further comprising:

a pump, coupled to the output of said detector, for providing negative pressure to the apparatus;

a proportionating valve, coupled to said sample input, for adjusting the concentration level of said test compound transferred to said mixing zone from said one or more sample inputs, wherein said one or more sample inputs further comprise;

a first intake nozzle for receiving each of said specified test compounds;

a second intake nozzle for receiving a buffer solution; and

the proportionating valve receives specified concentrations of each of the specified test compounds by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.

56. The apparatus of claim 43 further comprising a plurality of cell suspension reservoirs.

57. The apparatus of claim 43 further comprising a standard compound sampler, coupled to said mixing zone, for providing one or more standard compounds having a known effect on said cellular response of said suspended cells or a known interaction with said particles, wherein said mixing zone receives said one or more standard compounds from the standard compound sampler and mixes said one or more standard compounds with the suspended cells or particles and said detector measures the cellular response of the suspended cells to said one or more standard compounds or the interaction between said one or more standard compounds and said particles.

58. The apparatus of claim 57 wherein said mixing zone simultaneously mixes said sample and said one or more standard compounds with said suspended cells or said particles and said detector detects said known effect or an alteration of said known effect on the cellular responses of said suspended cells or said known interaction between said standard compound and said particles or an alteration of said known interaction between said standard compound and said particles.

59. The apparatus of claim 58 wherein said standard compound sampler is an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds.

66. The apparatus of claim 42, wherein said test compound source is capable of providing said test compounds at a single concentration or over a range of concentrations.

67. The apparatus of claim 42, further comprising a known compound source capable of sequentially providing each of a plurality of compounds which produce a known cellular response, said known compound source being in fluid communication with said mixing chamber such that said mixing chamber can combine a test compound, a compound which produces a known cellular response, and a cell suspension.

68. The apparatus of claim 67, wherein said known compound source is capable of providing the compounds which produce a known cellular response at a single concentration or over a range of concentrations.

60. An apparatus comprising:

one or more automated sample inputs for sequentially providing each of a plurality of samples, said each of said samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;

one or more automated cell or particle inputs for sequentially providing each of a plurality of cell suspensions or particles;

a mixing zone, coupled to said sample input, for receiving the samples, receiving the cell suspension or particles from the cell or particle input and mixing each sample with the cell suspensions or particles; and

a detector which detects one or more signals from a single cell or particle, said detector being coupled to the mixing zone and being capable of measuring one or more cellular responses in the suspended cells or determining whether one or more molecules are present in said samples.

61. The apparatus of claim 60, further comprising a coupler disposed between said mixing zone and said detector.

62. The apparatus of claim 61, wherein said coupler delivers slugs comprising said samples and said cell suspension or particles to said detector.

63. The apparatus of claim 62, wherein said coupler delivers substantially undiluted slugs to said detector.

64. The apparatus of claim 60 further comprising an input system for inputting solutions into said mixing zone.

65. The apparatus of claim 60, wherein said detector delivers cells having a desired phenotype or cellular response to a receptacle.

69. The apparatus of claim 60, wherein said one or more sample inputs is capable of providing said test compounds at a single concentration or over a range of concentrations.

70. The apparatus of claim 60, further comprising a known compound source capable of sequentially providing each of a plurality of compounds which produce a known cellular response, said known compound source being in fluid communication with said mixing zone such that said mixing zone can combine a test compound, a compound which produces a known cellular response, and a cell suspension.

71. The apparatus of claim 70, wherein said known compound source is capable of providing the compounds which produce a known cellular response at a single concentration or over a range of concentrations.